De novo 2.3 Mb microdeletion of 1q32.2 involving the Van der Woude Syndrome locus
© Tan et al.; licensee BioMed Central Ltd. 2013
Received: 2 May 2013
Accepted: 4 June 2013
Published: 6 August 2013
Van der Woude syndrome is the most common among syndromes which include cleft lip and/or cleft palate as one of the presentations. It is usually caused by mutations in the interferon regulatory factor 6 (IRF6) gene.
We previously reported on a patient with suspected deletion of the IRF6 gene. Using the Affymetrix Human SNP 6.0 Array, the interstitial deletion has been confirmed and found to be approximately 2.327–2.334 Mb within the 1q32.2 region. Although several known genes were deleted, the patient has no other phenotype apart from the orofacial presentations typical of VWS. The same deletion was not present in either parent and his two siblings were also phenotypically normal.
Other than IRF6, the genes which are deleted in this patient appear to be insensitive to copy number and haploinsufficiency. We compared the deletion in this patient with another case which was also mapped by high resolution array but had additional phenotypic features.
Keywords1q32 IRF6 gene Microdeletion Orofacial clefting SNP array Syndromic clefting Van der Woude syndrome
Cleft lip and/or cleft palate are common congenital birth defects which can occur in isolation or as part of a syndromic disorder. Among the more than 300 syndromes with orofacial clefting as one of the associated features, Van der Woude syndrome (VWS; MIM #119300) is the most common, accounting for approximately 2% of all cases. Except for the presence of paramedian lower lip pits and hypodontia, the presentation closely resembles that of isolated cleft lip and/or cleft palate. The inheritance pattern is autosomal dominant with the frequency at approximately one in 35,000 –100,000.
In 2002, the gene involved in VWS was identified as that encoding the interferon regulatory factor 6 (IRF6), a member of the interferon regulatory factor family of transcription factors . The study identified 46 mutations in IRF6 in patients with VWS and another 13 in patients with popliteal pterygium syndrome (PPS; MIM 119500). The PPS phenotype includes other congenital anomalies such as webbing of the skin, bifid scrotum, syndactyly of the fingers or toes in addition to orofacial clefting.
The two different syndromic disorders are caused by mutations in the same gene but the resulting phenotype depends on the exact nucleotide or amino acid involved and the position of the mutation. The mechanism is suggested to be haploinsufficiency for VWS and dominant-negative for PPS . Most of the identified mutations in VWS are nonsense and missense mutations found in exons which encode the DNA-binding or protein-binding domains. In the case of PPS, except for one nonsense mutation (Q393X), the rest involve substitutions of amino acid residues in the DNA-binding domain which makes direct contact with DNA. In vitro binding assays with the IRF6 protein showed that the 12 of the 13 mutations identified in VWS/PPS patients and mapped within the DNA-binding domain inhibited DNA binding. For mutations within the protein-binding region, six out of seven inhibited transcriptional activation completely, while the remaining one had the opposite effect .
Mutations in IRF6 have been identified in VWS patients from different ethnic groups. Most cases of VWS are inherited. Although penetrance is incomplete, it is still high at approximately 92%. Sequence analysis of the IRF6 coding region (exons 1–9) can detect mutations in 70% of patients with the VWS phenotype, of which 80% of the mutations would be within the protein-coding exons 3–9. In less than 2% of individuals with VWS, the entire IRF6 gene is deleted .
We previously described a case of de novo deletion as observed by the loss of paternal alleles and complete homozygosity of IRF6 gene polymorphisms. The reduced gene dosage was confirmed by MLPA . In this paper, we described the mapping of the deletion in this patient using a high resolution single nucleotide polymorphism (SNP) array.
The patient is the eldest of three children of healthy unrelated parents of Chinese ancestry. There was no significant family history of cleft lip and palate. He was previously found to have features consistent with Van der Woude Syndrome due to the presence of cleft lip and palate and lower lip pits.
DNA was extracted from frozen whole blood samples using the Gentra Puregene Blood Kit (Gentra Systems Inc., Minneapolis, USA). It was checked for quantity and purity using the NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, USA). Genome-wide Human SNP 6.0 Array (Affymetrix Inc., Santa Clara, USA) containing more than 906,600 SNPs and more than 946,600 copy number probes was used. Labeling, hybridization, washing, scanning and image extraction were performed by an Affymetrix certified service laboratory according to manufacturer’s instructions. Data was analyzed using Chromosome Analysis Suite.
Genes and microRNAs in the region deleted in the patient in this report and Salahshourifar et al. 2011
CR-1 like 3b/4b binding protein
CD46 antigen, complement regulatory protein
Homo sapiens cDNA FLJ41182 fis, clone BRACE2043349
Hypothetical gene – non-coding RNA
CD34 antigen isoform b
Hypothetical protein LOC642587
Calcium/calmodulin-dependent protein kinase IG
Laminin, beta 3 precursor
11-beta-hydroxysteroid dehydrogenase 1
TRAF3-interacting JNK-activating modulator
Chromosome 1 open reading frame 74; hypothetical protein LOC148304
Interferon regulatory factor 6
C1orf107; (digestive-organ expansion factor homolog)
SERTAD4 antisense RNA 1 (SERTAD4-AS1)
SERTA domain containing 4
Potassium voltage-gated channel, subfamily H (eag-related), member 1
Full-length cDNA clone CS0DJ006YN03 of T cells
REST corepressor 3 isoform d
Homo sapiens mRNA for KIAA1343 protein, partial cds
TNF receptor-associated factor 5
Homo sapiens cDNA FLJ27347
C1orf97;Homo sapiens long intergenic non-protein coding RNA 467
Retinal degeneration 3
Solute carrier family 30 (zinc transporter) member 1
Full-length cDNA clone CS0DK012YI08 of HeLa cells
NIMA-related kinase 2
Lysophosphatidylglycerol acyltransferase 1
Integrator complex subunit 7
Homo sapiens microRNA 3122
Protein phosphatase 2, regulatory subunit B
Homo sapiens FKSG56 (FKSG56) mRNA
Homo sapiens small nucleolar RNA, H/ACA box 16B
Transmembrane protein 206
Neuron derived neurotrophic factor precursor
The VWS locus was first mapped to the chromosomal region 1q32-41  before mutations in the IRF6 gene were identified in patients with VWS and PPS . Although SNPs in the gene have been associated with non-syndromic cleft lip and/or cleft palate , no other syndrome has been linked to the gene. While most identified mutations in VWS families were single nucleotide substitutions, there are a few cases of deletions. Most of the latter were small within-gene deletions which ranged from 5 to18 bp . There was one report of a 17-kb deletion involving exons 4–9 in a Japanese family .
Summary of VWS cases with deletion ≥ 1 Mb
Bocian & Walker 
Facial dysmorphism, skeletal abnormality, hypotonia
~ 1 Mb
Salahshourifar et al. 
Dysmorphism, growth retardation
In the present case, the breakpoint for the proximal end of the deletion is within a segment known as variation_3328 which is a copy number polymorphism (CNV). The distal breakpoint is within a large intron of the STY14 gene. Some of the genes in the deleted region are associated with conditions listed in Online Mendelian Inheritance in Men (OMIM). They are CR1L with SLE susceptibility, CD46 with measles, LAMB3 with epidermolysis bullosa (OMIM #226650 and 226700), and HSD11B1 with cortisone reductase deficiency. However, this patient has no other clinically significant pathology. There is no psychomotor delay or intellectual disability commonly found in patients with microdeletions involving multiple genes, therefore it appears that the other deleted genes are not sensitive to copy number changes. Indeed this is consistent with the scores for Hapoinsufficiency Index (HI) according to the DECIPHER database . The IRF6 gene which has the most significant HI index is also the only gene which could be linked to the patient’s phenotype.
Developmental delay and dysmorphism was reported for Case 1. Family VWS 1473 (Case 2) involved affected members over three generations . This is the only family with developmental and psychomotor delay out of over 300 VWS families studied. Along with cleft lip/palate and lip pits, all affected relatives exhibited various forms of developmental delay. There is one other report of a child with VWS features and also presenting with mental retardation but there was no karyotype information . Segregation of the VWS phenotype with intellectual disability in these three instances suggests that there is a gene involved in cognitive development in the region, and it is due to a dominant mutation and not haploinsufficiency. The 2.3 Mb microdeletion in our patient is bigger than that found in VWS1473 (Case 2 in Table 2) but he has normal intelligence. He has been followed up closely for the last 20 years and there is no evidence of other clinically significant condition. The loss of so many genes with no additional phenotypic consequence other than VWS at birth is surprising but is consistent with studies showing that the other genes deleted are unlikely to be haploinsufficient.
The largest deletion reported thus far is 2.98 Mb (Case 4 in Table 2) detected using an Agilent 400 K CGH array . At the time of the report, the 22-month old child was meeting developmental milestones with no evidence of developmental delay. There were dysmorphic features (including syndactyly also seen in PPS) and some indication of growth retardation. This deletion is distinct from others in that both proximal and distal breakpoints are different from previously reported cases. The only deleted genes shared are CAMK1G, G0S2, TRAF31P3, and IRF6. The distal end extends much further and includes at least 10 more genes, three of which had Haploinsufficiency Index (HI index) of less than 10, indicating that they are dosage sensitive and expected to have phenotypic effect (Table 1). However, none of the three genes have been linked to the dysmorphic features observed in this patient. Interestingly, the Development Disorder Genotype-Phenotype Database (DDG2P) lists IRF6 as one of the genes associated with developmental disorders . It is the only gene within the deleted which is listed as having evidence of developmental delay in multiple cases. However, there is no evidence of developmental delay for both our patient and the patient with the 2.98 Mb deletion.
The deletion in our patient appeared to be a very rare event with only two other de novo cases reported. Our data suggest that other than IRF6, the genes that were deleted were not affected by haploinsufficiency.
Approval to conduct the study was granted by the SingHealth Institutional Review Board. Written informed consent was obtained from the patients’ parents.
1Principal Scientist (ECT) and Senior Medical Technologist (ECPL), KK Research Centre, KK Women’s and Children’s Hospital, 100 Bukit Timah Road, Singapore 229899. 2Adjunct Associate Professor, Office of Clinical Sciences, Duke-NUS Graduate Medical School Singapore, 8 College Road, Singapore 169857. 3Emeritus Consultant, Department of Plastic, Reconstructive & Aesthetic Surgery, Singapore General Hospital, Outram Road, Singapore 169608.
Bacterial artificial chromosome
Copy number variant
Mendelian inheritance in Men
Popliteal pterygium syndrome
Single nucleotide polymorphism
Van der Woude syndrome.
We are grateful to the patient and his parents for participating in this study. This work was supported by project grant KRAU151/09 from KK Women’s and Children’s Hospital and NMRC/PPG/KKH12010-Theme3 from the National Medical Research Council, Ministry of Health, Republic of Singapore.
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